The objective of this proposal is to clarify the process by which cutaneous contact with reactive haptens leads to activation of the immune system by developing in assays that will employ epidermal Langerhans cells (LC) to study the requirements for, and factors influencing the, activation and proliferation of unprimed and primed, hapten-specific T lymphocytes at the clonal level. A major working hypothesis is that activation of an unprimed T cell population by relevant antigen presenting cells (APC) will define the frequency of T cell precursors for a given set of antigens, thus providing a measure of immunogenicity as well as APC efficacy. The specific aims are: 1) To develop an in vitro model of contact hypersensitivity (CH) using clonal activation of unprimed T cells by hapten-conjugated LC in which the principal methodology is limiting dilution microculture of T cells from peripheral lymph nodes and spleens of nonsensitized mice stimulated at clonal levels by syngeneic LC conjugated to DNFB or to FITC. 2) To identify the antigens in CH to DNFB and FITC by analyzing the proliferative T cell precursor frequencies that result from cocultures of LC and unprimed T cells in which hapten is provided in conjugated form to either LC, keratinocytes, cell membrane preparations, or specific proteins. 3) To define mechanisms of processing and presentation of immunogenic forms of haptens by LC using: immunofluorescence microscopy and FACS analysis to track routing of antigen within LC, inhibition of sequential steps in antigen processing, and antigen presentation-blocking experiments using mAb against specific accessory molecules. 4) To characterize the T cell clones that proliferate following primary and secondary activation in vitro based on surface phenotype, lymphokine synthesis, antigen and APC requirements, accessory needs for growth and activation, and the capacity to induce or suppress CH when adoptively transferred into naive animals. If successful, these in vitro assays will serve as prototypes for the development of similar tests in humans to screen for, and assess the effects of, contact sensitizers.